The platelet-derived growth factor receptor (PDGFR) is a membrane tyrosine kinase receptor involved in several metabolic pathways, not only physiological but also pathological, as in tumor ...progression, immune-mediated diseases, and viral diseases. Considering this macromolecule as a druggable target for modulation/inhibition of these conditions, the aim of this work was to find new ligands or new information to design novel effective drugs. We performed an initial interaction screening with the human intracellular PDGFRα of about 7200 drugs and natural compounds contained in 5 independent databases/libraries implemented in the MTiOpenScreen web server. After the selection of 27 compounds, a structural analysis of the obtained complexes was performed. Three-dimensional quantitative structure-activity relationship (3D-QSAR) and absorption, distribution, metabolism, excretion, and toxicity (ADMET) analyses were also performed to understand the physicochemical properties of identified compounds to increase affinity and selectivity for PDGFRα. Among these 27 compounds, the drugs Bafetinib, Radotinib, Flumatinib, and Imatinib showed higher affinity for this tyrosine kinase receptor, lying in the nanomolar order, while the natural products included in this group, such as curcumin, luteolin, and epigallocatechin gallate (EGCG), showed sub-micromolar affinities. Although experimental studies are mandatory to fully understand the mechanisms behind PDGFRα inhibitors, the structural information obtained through this study could provide useful insight into the future development of more effective and targeted treatments for PDGFRα-related diseases, such as cancer and fibrosis.
Lung fibrosis is a severe condition resulting from several interstial lung diseases (ILD) with different etiologies. Current therapy of ILD, especially those associated with connective tissue ...diseases, is rather limited and new anti-fibrotic strategies are needed. In this study, we investigated the anti-fibrotic activity in vivo of human mesenchymal stromal cells obtained from whole umbilical cord (hUC-MSC). Adult immunocompetent C57BL/6 mice (n. = 8 for each experimental condition) were injected intravenously with hUC-MSC (n. = 2.5 × 105) twice, 24 hours and 7 days after endotracheal injection of bleomycin. Upon sacrifice at days 8, 14, 21, collagen content, inflammatory cytokine profile, and hUC-MSC presence in explanted lung tissue were analyzed. Systemic administration of a double dose of hUC-MSC significantly reduced bleomycin-induced lung injury (inflammation and fibrosis) in mice through a selective inhibition of the IL6-IL10-TGFβ axis involving lung M2 macrophages. Only few hUC-MSC were detected from explanted lungs, suggesting a "hit and run" mechanism of action of this cellular therapy. Our data indicate that hUC-MSC possess strong in vivo anti-fibrotic activity in a mouse model resembling an immunocompetent human subject affected by inflammatory ILD, providing proof of concept for ad-hoc clinical trials.
Objective
To describe a skin–SCID mouse chimeric model of systemic sclerosis (SSc; scleroderma) fibrosis based on engraftment of ex vivo–bioengineered skin using skin cells derived either from ...scleroderma patients or from healthy donors.
Methods
Three‐dimensional bioengineered skin containing human keratinocytes and fibroblasts isolated from skin biopsy specimens from healthy donors or SSc patients was generated ex vivo and then grafted onto the backs of SCID mice. The features of the skin grafts were analyzed by immunohistochemistry, and the functional profile of the graft fibroblasts was defined before and after treatment with IgG from healthy controls or SSc patients. Two procedures were used to investigate the involvement of platelet‐derived growth factor receptor (PDGFR): 1) nilotinib, a tyrosine kinase inhibitor, was administered to mice before injection of IgG from SSc patient sera (SSc IgG) into the grafts, and 2) human anti‐PDGFR monoclonal antibodies were injected into the grafts.
Results
Depending on the type of bioengineered skin grafted, the regenerated human skin exhibited either the typical scleroderma phenotype or the healthy human skin architecture. Treatment of animals carrying healthy donor skin grafts with SSc IgG resulted in the appearance of a bona fide scleroderma phenotype, as confirmed by increased collagen deposition and fibroblast activation markers. Results of the experiments involving administration of nilotinib or monoclonal antibodies confirmed the involvement of PDGFR.
Conclusion
Our results provide the first in vivo demonstration of the fibrotic properties of anti‐PDGFR agonistic antibodies. This bioengineered skin–humanized mouse model can be used to test in vivo the progression of the disease and to monitor response to antifibrotic drugs.
Systemic sclerosis (scleroderma) is characterized by immunologic abnormalities, injury of endothelial cells, and tissue fibrosis. Abnormal oxidative stress has been documented in scleroderma and ...linked to fibroblast activation. Since platelet-derived growth factor (PDGF) stimulates the production of reactive oxygen species (ROS) and since IgG from patients with scleroderma reacts with human fibroblasts, we tested the hypothesis that patients with scleroderma have serum autoantibodies that stimulate the PDGF receptor (PDGFR), activating collagen-gene expression.
We analyzed serum from 46 patients with scleroderma and 75 controls, including patients with other autoimmune diseases, for stimulatory autoantibodies to PDGFR by measuring the production of ROS produced by the incubation of purified IgG with mouse-embryo fibroblasts carrying inactive copies of PDGFR alpha or beta chains or the same cells expressing PDGFR alpha or beta. Generation of ROS was assayed with and without specific PDGFR inhibitors. Antibodies were characterized by immunoprecipitation, immunoblotting, and absorption experiments.
Stimulatory antibodies to the PDGFR were found in all the patients with scleroderma. The antibodies recognized native PDGFR, inducing tyrosine phosphorylation and ROS accumulation. Autoantibody activity was abolished by preincubation with cells expressing the PDGFR alpha chain or with recombinant PDGFR or by PDGFR tyrosine kinase inhibitors. Stimulatory PDGFR antibodies selectively induced the Ha-Ras-ERK1/2 and ROS cascades and stimulated type I collagen-gene expression and myofibroblast phenotype conversion in normal human primary fibroblasts.
Stimulatory autoantibodies against PDGFR appear to be a specific hallmark of scleroderma. Their biologic activity on fibroblasts strongly suggests that they have a causal role in the pathogenesis of the disease.
•Agonistic antibodies activate pathways leading to tissue and vascular damage in SSc.•Anti-endothelial cells antibodies (AECA) can induce either apoptosis or activation.•Antibodies targeting specific ...PDGFRα epitopes can induce skin fibrosis in vivo.•Anti-AT1R/ETAR antibodies are associated with severe SSc vascular manifestations.•Anti-muscarinic-3 receptor (M3R) antibodies may cause gastrointestinal dysfunction.
Systemic sclerosis (SSc) is characterized by microangiopathy, excessive fibrosis, and the presence of circulating autoantibodies to several cellular and extracellular components. The role of autoimmunity in generating the clinical and pathologic phenotypes in SSc has been long debated and is still matter of controversy. Distinct specificities of antinuclear antibodies (ANAs) are selectively detected in SSc patients and are associated with unique disease manifestations, but do not have a proven pathogenic role. A new group of autoantibodies reactive with cell surface receptors have been identified in SSc patients. They have been shown to directly activate pathways that may contribute to tissue and vascular damage. As such, they are proposed to have a role as agonistic autoantibodies in SSc. According to Koch’s third postulate, the autoantibodies in question should cause disease when introduced into a healthy subject. Therefore, our review will focus on those autoantibodies for which agonistic activity has already been demonstrated not only in vitro, but, at least partly, also in vivo. These include the antibodies anti-endothelial cells (AECA), anti-Platelet-Derived Growth Factor Receptor (PDGFR), anti-Angiotensin II type 1 receptor (AT1R) and anti-endothelin-1 type A receptor (ETaR). In this review, we will discuss also a class of antagonistic autoantibodies, the anti-muscarinic-3 receptor (M3R) antibodies, since they seem to fulfill the aforementioned requirements.
Purpose
The most harmful atmospheric pollutant for human health is particulate matter (PM). We analyzed the correlation between short-term lag exposure to PM10 and PM2.5, salivary cortisol and TNF-α ...level, and methylation levels of the TNF-α promoter.
Methods
A pilot study including 20 subjects. Eight salivary samples for each subject at various times of the day were collected for comparing cortisol levels and TNFα detection. TNFα promoter methylation levels on salivary DNA were analyzed. Regression analyses were performed using generalized linear mixed models between the different outcomes and 4, 3, 2 and 1 day’s lag values of PM10/PM2.5.Generalized additive mixed model (GAMM) was used to evaluate any potential deviation from linearity.
Results
Area under the curve with respect to the ground (AUCg) showed a statistically positive association with 4-, 3-, 2-, and 1-day lag of exposure to PM10. Area under the curve with respect to the increase (AUCi) showed a statistically negative association with 4-, 3- and 1-day lag of exposure to PM10. TNFα showed statistically significant association with both exposures, PM10 and PM2.5, at 4-, 3-, 2-, and 1-day lag.
Conclusions
Regarding cortisol levels there is an increase of overall hormone levels but a less dynamism of the system to answer to external stressors. Increase of TNF-α may reflect increased levels of oxidative stress and inflammation due to pollution exposure.
Objective
Reactive oxygen species (ROS) contribute to the pathogenesis of fibrosis in systemic sclerosis (SSc; scleroderma), and NADPH oxidase (NOX) is an important source of ROS. Since the role of ...single NOX isoforms has not been previously investigated in SSc, this study was undertaken to assess the expression of NOX in SSc fibroblasts compared to normal healthy cells and to analyze their role in cell activation.
Methods
Expression of NOX isoforms in dermal fibroblasts from patients with SSc and healthy control subjects was analyzed by real‐time polymerase chain reaction, immunoblotting, and immunofluorescence. NOX isoforms were silenced using small interfering RNA. Production of ROS was measured by fluorometry and confocal microscopy.
Results
Scleroderma fibroblasts showed up‐regulation of NOX‐2 and NOX‐4 protein and messenger RNA (mRNA) expression. Treatment of the cells with diphenyleneiodonium, a nonselective inhibitor of flavin‐containing enzymes, and silencing of NOX2 and NOX4 decreased the production of ROS as well as the expression of type I collagen and α‐smooth muscle actin in SSc fibroblasts. ROS generated by NOX‐2 and NOX‐4 were involved in DNA damage and activation of a DNA repair checkpoint. Incubation of healthy control fibroblasts with platelet‐derived growth factor (PDGF) or with IgG isolated from SSc patient serum enhanced the expression of NOX2 and NOX4 mRNA, via ROS, in a time‐dependent manner. Treatment with actinomycin D, a transcription inhibitor, reversed the effects of PDGF stimulation but not the effects of SSc IgG.
Conclusion
Both NOX2 and NOX4 generate ROS in SSc fibroblasts and play a critical role in cell activation and DNA damage. Expression of NOX‐2 and NOX‐4 in SSc fibroblasts is maintained by a ROS‐mediated loop.
Systemic sclerosis (scleroderma, SSc) is a systemic disease characterized by vascular lesions, fibrosis, and circulating autoantibodies. A complex interplay between innate and adaptive immunity, and ...with regard to the latter, between humoral and cellular immunity, is believed to be involved in SSc pathogenesis. Lately, close attention has been paid to the role of B cells which, once activated, release profibrotic cytokines, promote profibrotic Th2 differentiation, and produce autoantibodies. Several novel interesting autoantibodies, targeting antigens within the extracellular matrix or on the cell surface, rather than the nuclear antigens of canonical SSc-autoantibodies, have been recently described in patients with SSc. As they show stimulatory or inhibitory activity or react with structures involved in the pathogenesis of SSc lesions, they can be considered as potentially pathogenic. In this paper, we will review those which have been better characterized.
The stratification of mortality risk in COVID-19 patients remains extremely challenging for physicians, especially in older patients. Innovative minimally invasive molecular biomarkers are needed to ...improve the prediction of mortality risk and better customize patient management. In this study, aimed at identifying circulating miRNAs associated with the risk of COVID-19 in-hospital mortality, we analyzed serum samples of 12 COVID-19 patients by small RNA-seq and validated the findings in an independent cohort of 116 COVID-19 patients by qRT-PCR. Thirty-four significantly deregulated miRNAs, 25 downregulated and 9 upregulated in deceased COVID-19 patients compared to survivors, were identified in the discovery cohort. Based on the highest fold-changes and on the highest expression levels, 5 of these 34 miRNAs were selected for the analysis in the validation cohort. MiR-320b and miR-483-5p were confirmed to be significantly hyper-expressed in deceased patients compared to survived ones. Kaplan-Meier and Cox regression models, adjusted for relevant confounders, confirmed that patients with the 20% highest miR-320b and miR-483-5p serum levels had three-fold increased risk to die during in-hospital stay for COVID-19. In conclusion, high levels of circulating miR-320b and miR-483-5p can be useful as minimally invasive biomarkers to stratify older COVID-19 patients with an increased risk of in-hospital mortality.
•Innovative biomarkers to stratify older patients with COVID-19 at high risk of in-hospital death are urgently needed.•Thirty-four miRNAs differentially expressed between deceased and surviving COVID-19 patients were identified through small RNA-seq analysis.•MiR-320b and miR-483-5p were confirmed to be significantly hyper-expressed in deceased patients compared to surviving ones.•Patients with serum miR-320b and miR-483-5p in the upper quintile had three-fold increased risk to die during in-hospital stay for COVID-19.
Abnormal oxidative stress has been described in systemic sclerosis (SSc) and previous works from our laboratory demonstrated an increased generation of reactive oxygen species (ROS) by SSc ...fibroblasts and monocytes. This study investigated the ability of SSc T lymphocytes to produce ROS, the molecular pathway involved, and the biological effects of ROS on SSc phenotype.
Peripheral blood T lymphocytes were isolated from serum of healthy controls or SSc patients by negative selection with magnetic beads and activated either with PMA or with magnetic beads coated with anti-CD3 and anti-CD28 antibodies. Intracellular ROS generation was measured using a DCFH-DA assay in a plate reader fluorimeter or by FACS analysis. CD69 expression and cytokine production were analyzed by FACS analysis. Protein expression was studied using immunoblotting techniques and mRNA levels were quantified by real-time PCR. Cell proliferation was carried out using a BrdU incorporation assay.
Peripheral blood T lymphocytes from SSc patients showed an increased ROS production compared to T cells from healthy subjects. Since NADPH oxidase complex is involved in oxidative stress in SSc and we found high levels of gp91phox in SSc T cells, SSc T cells were incubated with chemical inhibititors or specific siRNAs against gp91phox. Inhibition of NADPH oxidase partially reverted CD69 activation and proliferation rate increase, and significantly influenced cytokine production and ERK1/2 activation.
SSc T lymphocityes are characterized by high levels of ROS, generated by NADPH oxidase via ERK1/2 phosphorylation, that are essential for cell activation, proliferation, and cytokine production. These data confirm lymphocytes as key cellular players in the pathogenesis of systemic sclerosis and suggest a crucial link between ROS and T cell activation.
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